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PDZD8 Disruption Causes Cognitive Impairment in Humans, Mice, and Fruit Flies

Open AccessPublished:January 10, 2022DOI:https://doi.org/10.1016/j.biopsych.2021.12.017

      Abstract

      Background

      The discovery of coding variants in genes that confer risk of intellectual disability (ID) is an important step toward understanding the pathophysiology of this common developmental disability.

      Methods

      Homozygosity mapping, whole-exome sequencing, and cosegregation analyses were used to identify gene variants responsible for syndromic ID with autistic features in two independent consanguineous families from the Arabian Peninsula. For in vivo functional studies of the implicated gene’s function in cognition, Drosophila melanogaster and mice with targeted interference of the orthologous gene were used. Behavioral, electrophysiological, and structural magnetic resonance imaging analyses were conducted for phenotypic testing.

      Results

      Homozygous premature termination codons in PDZD8, encoding an endoplasmic reticulum–anchored lipid transfer protein, showed cosegregation with syndromic ID in both families. Drosophila melanogaster with knockdown of the PDZD8 ortholog exhibited impaired long-term courtship-based memory. Mice homozygous for a premature termination codon in Pdzd8 exhibited brain structural, hippocampal spatial memory, and synaptic plasticity deficits.

      Conclusions

      These data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in a neurodevelopmental cognitive disorder. Model organisms with manipulation of the orthologous gene replicate aspects of the human phenotype and suggest plausible pathophysiological mechanisms centered on disrupted brain development and synaptic function. These findings are thus consistent with accruing evidence that synaptic defects are a common denominator of ID and other neurodevelopmental conditions.

      Keywords

      Intellectual disability (ID) refers to a heterogeneous group of neurodevelopmental disorders affecting 2% to 3% of the general population, characterized by significant impairment in cognitive ability and adaptive behaviors. It is usually subdivided into nonsyndromic and syndromic forms, depending on the manifestation of additional physical, neurologic, and/or metabolic abnormalities. Typically identified in childhood because of delayed developmental milestones, affected individuals struggle with memory, problem solving, language, and visual comprehension, reflected by an IQ score of <70 (
      American Psychiatric Association
      Diagnostic and Statistical Manual of Mental Disorders.
      ).
      ID has high phenotypic variability and etiologic diversity. Based on the IQ score, ID can be classified as mild (50–69), moderate (35–49), severe (20–34), or profound (under 20) (
      World Health Organization
      The ICD-10 Classification of Mental and Behavioural Disorders: Clinical Descriptions and Diagnostic Guidelines.
      ). Among the known causes, approximately 50% of ID cases have an early environmental etiology, such as intrauterine exposure to alcohol, the most common nonheritable cause of ID (
      • O’Leary C.
      • Leonard H.
      • Bourke J.
      • D’Antoine H.
      • Bartu A.
      • Bower C.
      Intellectual disability: Population-based estimates of the proportion attributable to maternal alcohol use disorder during pregnancy.
      ). The remaining ∼50% of ID cases of known cause have a genetic etiology, such as chromosomal abnormalities or mutations in specific genes (
      • Vissers L.E.L.M.
      • Gilissen C.
      • Veltman J.A.
      Genetic studies in intellectual disability and related disorders.
      ).
      Because ID negatively affects fecundity, dominant autosomal variants occurring de novo may contribute to a large proportion of sporadic cases, particularly in outbred Western populations (
      • Gilissen C.
      • Hehir-Kwa J.Y.
      • Thung D.T.
      • van de Vorst M.
      • van Bon B.W.M.
      • Willemsen M.H.
      • et al.
      Genome sequencing identifies major causes of severe intellectual disability.
      ). Autosomal recessive variants play a significant role in ID in populations with frequent parental consanguinity, such as in the Middle East (
      • Morton N.E.
      Effect of inbreeding on IQ and mental retardation.
      ,
      • Mir Y.R.
      • Kuchay R.A.H.
      Advances in identification of genes involved in autosomal recessive intellectual disability: A brief review.
      ). Defects in more than 700 genes have been implicated in ID, and a significant overlap has been noted with genes identified in other neurodevelopmental disorders such as autism spectrum disorder (ASD) (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      ). Functional categorization of the encoded proteins has revealed significant enrichment of proteins involved in glutamatergic synapse structure and function (
      • Volk L.
      • Chiu S.L.
      • Sharma K.
      • Huganir R.L.
      Glutamate synapses in human cognitive disorders.
      ,
      • Torres V.I.
      • Vallejo D.
      • Inestrosa N.C.
      Emerging synaptic molecules as candidates in the etiology of neurological disorders.
      ,
      • Moretto E.
      • Murru L.
      • Martano G.
      • Sassone J.
      • Passafaro M.
      Glutamatergic synapses in neurodevelopmental disorders.
      ). Despite the considerable progress in understanding, no treatment is currently available for ID, and at least 50% of the estimated genetic causes of ID remain unknown (
      • Willemsen M.H.
      • Kleefstra T.
      Making headway with genetic diagnostics of intellectual disabilities.
      ).
      Herein, we report the clinical features and molecular diagnosis of two independent consanguineous families affected by syndromic ID from the Arabian Peninsula. Through the application of homozygosity mapping and whole-exome sequencing (WES), we report that all affected individuals are homozygous for premature termination codons (PTCs) in PDZD8 (formerly PDZK8), a gene of five exons located at 10q25.3-q26.11, encoding a 1154 aa endoplasmic reticulum (ER) transmembrane (TM) protein.
      In neurons, depletion of PDZD8 has been shown to impair endosomal homeostasis (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ), decrease the proximity of the ER and mitochondria (
      • Hertlein V.
      • Flores-Romero H.
      • Das K.K.
      • Fischer S.
      • Heunemann M.
      • Calleja-Felipe M.
      • et al.
      MERLIN: A novel BRET-based proximity biosensor for studying mitochondria-ER contact sites.
      ), and decrease calcium ion (Ca2+) uptake by mitochondria following synaptic transmission–induced Ca2+ release from the ER (
      • Hirabayashi Y.
      • Kwon S.K.
      • Paek H.
      • Pernice W.M.
      • Paul M.A.
      • Lee J.
      • et al.
      ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.
      ).
      Because assessing human gene function in cognition is challenging, we used a cross-species approach. We report that targeted interference of the PDZD8 orthologs in fruit flies and mice leads to long-term memory, brain structural, and synaptic plasticity deficits. Our findings are consistent with accruing evidence that glutamatergic synapse dysfunction represents a common underlying pathogenic mechanism in ID and other neurodevelopmental disorders (
      • Kochinke K.
      • Zweier C.
      • Nijhof B.
      • Fenckova M.
      • Cizek P.
      • Honti F.
      • et al.
      Systematic phenomics analysis deconvolutes genes mutated in intellectual disability into biologically coherent modules.
      ,
      • Volk L.
      • Chiu S.L.
      • Sharma K.
      • Huganir R.L.
      Glutamate synapses in human cognitive disorders.
      ,
      • Torres V.I.
      • Vallejo D.
      • Inestrosa N.C.
      Emerging synaptic molecules as candidates in the etiology of neurological disorders.
      ).

      Methods and Materials

      For more detailed methodology, see the Supplemental Experimental Procedures.

      Ethical Approvals

      The human study was approved by the Sultan Qaboos University Ethical Committee. Informed consent was obtained from the parents of the affected individuals using a process that adhered to the tenets of the Declaration of Helsinki. The mouse study was conducted in accordance with the UK Animals (Scientific Procedures) Act 1986 under UK Home Office licenses and approved by institutional Animal Welfare and Ethical Review Bodies.

      Sequencing and Variant Identification

      Homozygosity mapping and WES were conducted as described previously (
      • Al-Amri A.
      • Al Saegh A.
      • Al-Mamari W.
      • El-Asrag M.E.
      • Ivorra J.L.
      • Cardno A.G.
      • et al.
      Homozygous single base deletion in TUSC3 causes intellectual disability with developmental delay in an Omani family.
      ). Segregation in families was confirmed by polymerase chain reaction and Sanger sequencing.

      Drosophila melanogaster

      The UAS-CG10362-RNAi (v.8317; UAS-RNAi) line (
      • Dietzl G.
      • Chen D.
      • Schnorrer F.
      • Su K.C.
      • Barinova Y.
      • Fellner M.
      • et al.
      A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.
      ) was crossed with the Act5C-Gal4 ubiquitous driver line to induce ubiquitous expression of a specific 326-bp hairpin structure (CG10362-RNAi) that inhibits expression of the target CG10362 gene via RNA interference. Behavioral testing was performed as described previously (
      • McBride S.M.
      • Giuliani G.
      • Choi C.
      • Krause P.
      • Correale D.
      • Watson K.
      • et al.
      Mushroom body ablation impairs short-term memory and long-term memory of courtship conditioning in Drosophila melanogaster.
      ,
      • Mery F.
      • Kawecki T.J.
      A cost of long-term memory in Drosophila.
      ,
      • Koemans T.S.
      • Oppitz C.
      • Donders R.A.T.
      • van Bokhoven H.
      • Schenck A.
      • Keleman K.
      • Kramer J.M.
      Drosophila courtship conditioning as a measure of learning and memory.
      ).

      Mice

      C57BL/6NTac-Pdzd8tm1b(EUCOMM)Wtsi mice were generated by replacing an 835-bp sequence including exon 3 with a lacZ expression cassette, which created a frameshift that changed the phenylalanine (F) and isoleucine (I) at positions 333 and 334 to an asparagine (N) and a termination codon (∗) (p.F333Nfs1∗) (Figure S1A, C) (
      • Friedel R.H.
      • Seisenberger C.
      • Kaloff C.
      • Wurst W.
      EUCOMM—The European conditional mouse mutagenesis program.
      ). Heterozygotes were intercrossed to generate Pdzd8 homozygous mutant (Pdzd8tm1b; tm1b) and heterozygous and wild-type (WT) littermates for phenotypic testing.

      Mouse Behavioral Testing

      Behavioral testing of early adults over 8 weeks of age was performed as described previously (
      • Dachtler J.
      • Glasper J.
      • Cohen R.N.
      • Ivorra J.L.
      • Swiffen D.J.
      • Jackson A.J.
      • et al.
      Deletion of α-neurexin II results in autism-related behaviors in mice.
      ,
      • Kirshenbaum G.S.
      • Dachtler J.
      • Roder J.C.
      • Clapcote S.J.
      Characterization of cognitive deficits in mice with an alternating hemiplegia-linked mutation.
      ,
      • Illouz T.
      • Madar R.
      • Clague C.
      • Griffioen K.J.
      • Louzoun Y.
      • Okun E.
      Unbiased classification of spatial strategies in the Barnes maze.
      ,
      • Riedel G.
      • Robinson L.
      • Crouch B.
      Spatial learning and flexibility in 129S2/SvHsd and C57BL/6J mouse strains using different variants of the Barnes maze.
      ).

      Electrophysiology

      Extracellular field recordings using transverse hippocampal slices prepared from 4- to 6-week-old mice were performed as described previously (
      • Dennis S.H.
      • Pasqui F.
      • Colvin E.M.
      • Sanger H.
      • Mogg A.J.
      • Felder C.C.
      • et al.
      Activation of muscarinic M1 acetylcholine receptors induces long-term potentiation in the hippocampus.
      ). Three different long-term potentiation (LTP) induction protocols were used. Theta burst stimulation (TBS) consisted of 10 bursts at 5 Hz, where each burst consisted of five stimuli at 100 Hz. This was applied either once (1× TBS) or three times separated by 10 seconds (3× TBS). High-frequency stimulation (HFS) consisted of one burst of 100 stimuli at 100 Hz (1× HFS).

      Structural Magnetic Resonance Imaging

      For high-resolution structural magnetic resonance imaging, 16-week-old mice were terminally anesthetized and intracardially perfused. Samples were processed, imaged, and analyzed as described previously (
      • Gompers A.L.
      • Su-Feher L.
      • Ellegood J.
      • Copping N.A.
      • Riyadh M.A.
      • Stradleigh T.W.
      • et al.
      Germline Chd8 haploinsufficiency alters brain development in mouse.
      ). A linear model with genotype and sex as predictors was fitted to the absolute (mm3) and relative volume of every region independently and to every voxel independently in the brains of Pdzd8tm1b and WT mice, with a false discovery rate threshold of 1%.

      Results

      Clinical Features

      Family A consists of 3 affected (A.IV.1, A.IV.2, and A.IV.5) and 2 unaffected (A.IV.3 and A.IV.4) siblings born to consanguineous parents (first cousins) (A.III.1 and A.III.2) within an extended Omani pedigree (Figure 1A). Clinical examination revealed that all affected individuals have moderate to severe ID with autistic features, myopathy, and facial dysmorphism (myopathic face with orbital hypertelorism, malar flattening, open mouth, and high-arched palate). In addition, each affected sibling had other specific health problems, as detailed in Table 1. Both the father (A.III.2) and an unaffected male sibling (A.IV.3) had mild autistic features, and both parents (A.III.1 and A.III.2) had mild myopathy and reduced facial expression.
      Figure thumbnail gr1
      Figure 1Two families with PDZD8 mutations. (A) Pedigree of four-generation family A showing cosegregation of PDZD8 p.(S733∗) homozygosity with syndromic ID in 3 affected siblings (represented by filled symbols). (B) Pedigree of four-generation family B showing cosegregation of PDZD8 p.(Y298∗) homozygosity with syndromic ID in the affected individual (represented by filled symbol). Two progeny who died in utero are represented by small triangles. The numbers in generation III indicate brothers and sisters of the parents (B.III.1 and B.III.2). (C) Sanger sequence chromatograms showing the PDZD8 4-bp (AGTT) deletion (c.2197_2200del) identified in family A. (D) Sanger sequence chromatograms showing the PDZD8 nonsense mutation (c.894C>G) identified in family B. (E) Schematic diagram depicting domain structure and functions of PDZD8 in human (Q8NEN9; top), mouse (B9EJ80; middle), and Drosophila (Q9VYR9; bottom). The ER-TM domain (2–24 aa) and a region between the PDZ and phorbol-ester/diacylglycerol binding (C1) domains (466–797 aa) are required for interaction with protrudin (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ,
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ). The SMP domain is required for the formation of PDZD8 dimers or oligomers (
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ). The SMP and PDZ domains are required for the extraction of lipids from the ER to late endosomes and lysosomes (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ). The C1 domain is required for interaction with PS and PI4P associated with the late endosome/lysosome membrane (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Hammond G.R.V.
      • Machner M.P.
      • Balla T.
      A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi.
      ). The CC domain is required for interaction with Rab-7a (
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ,
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ,
      • Guillén-Samander A.
      • Bian X.
      • De Camilli P.
      PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes.
      ). Black horizontal lines indicate interactor binding sites; broken vertical red lines indicate the location of PTC (human: p.Y298∗ & p.S733∗; mouse: p.F333Nfs1∗). Numbering is from published sources (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ,
      UniProt Consortium
      UniProt: The universal protein Knowledgebase in 2021.
      ). C, carboxyl-terminus; CC, coiled-coil; ER, endoplasmic reticulum; ID, intellectual disability; N, amino-terminus; PR, proline-rich; PS, phosphatidylserine; PI4P, phosphatidylinositol 4-phosphate; PDZ, PSD-95/DlgA/ZO-1-like; PTC, premature termination codon; SMP, synaptotagmin-like mitochondrial lipid-binding; TM, transmembrane; UAE, United Arab Emirates.
      Table 1Clinical Features of Patients With Mutations in PDZD8
      CharacteristicFamily A Affected IndividualsFamily B Affected Individual
      A.IV.1A.IV.2A.IV.5B.IV.2
      ConsanguinityYesYesYesYes
      Ethnic OriginOmaniOmaniOmaniEmirati
      Genotype, Mat/Patp.(S733∗)/p.(S733∗); c.2197_2200del/c.2197_2200delp.(S733∗)/p.(S733∗); c.2197_2200del/c.2197_2200delp.(S733∗)/p.(S733∗); c.2197_2200del/c.2197_2200delp.(Y298∗)/p.(Y298∗); c.894C>G/c.894C>G
      SexMaleMaleFemaleMale
      Age, Years3025177
      Developmental DelayYesYesYesYes
      Intellectual DisabilityYes (severe)Yes (moderate)Yes (severe)Yes (moderate)
      Autistic FeaturesYesYes (mild)YesYes (mild)
      Facial DysmorphismYesYesYesYes
      Orbital HypertelorismYesYesYesYes
      MyopiaNoNoYesYes
      MyopathyYesYes (mild)YesNo
      EpilepsyNoYes (controlled)YesNo
      Congenital Heart DefectsNoNoYesNo
      Marfanoid HabitusYesNoYesNo
      Other Behavioral ProblemsNoYes (OCD)Yes (ADHD; insomnia)Yes (ADHD)
      Brain Scan FindingsNDNDHypoplasia of splenium of corpus callosumNormal
      Other FindingsNoNoAmblyopia, cleft palate, scoliosisBilateral ptosis, astigmatism, overlapping toes
      Nucleotide and residue numbering are based on NM_173791.5.
      ADHD, attention-deficit/hyperactivity disorder; Mat, maternal; ND, not determined; OCD, obsessive-compulsive disorder; Pat, paternal.
      Family B consists of 1 affected (B.IV.2) and 3 unaffected (B.IV.1, B.IV.3, and B.IV.4) siblings born to first cousin parents (B.III.1 and B.III.2) within an Emirati pedigree (Figure 1B). On clinical examination at 4 years of age, the affected male (B.IV.2) presented with delayed speech, moderate ID, mild autistic features (echolalia, jumping, hand flapping, lack of eye contact), attention deficit, dysmorphic features (low-set ears with simple helix, bilateral ptosis), and other specific health problems detailed in Table 1.

      Identification of PDZD8 Mutations

      The pedigree structures of families A and B suggested autosomal recessive transmission of a homozygous mutant allele from a common ancestor as the most likely explanation for syndromic ID in each family. In family A, homozygosity mapping using single nucleotide polymorphism array data in affected males A.IV.1 and A.IV.2 and variants extracted from WES in affected female A.IV.5 identified homozygous regions on chromosomes 6 (2.57 Mb), 10 (28.28 Mb), 13 (5.20 Mb), and 17 (8.38 Mb) shared by all three (Figure S2A).
      WES in subject A.IV.5 revealed 9032 variants in these shared homozygous regions. After filtering for rare variants predicted to be pathogenic followed by segregation analysis, only variants in ANKRD2 [NM_020349.4: c.982C>T; p.(R328W)] and PDZD8 [NM_173791.5: c.2197_2200del; p.(S733∗)] remained, both within the 28.28-Mb region on chromosome 10 (Figure 1A and Figure S2B).
      The PDZD8 c.2197_2200del variant deletes four base pairs in exon 5, introducing a frameshift and PTC (Figure 1C and Table 2) absent from gnomAD. ANKRD2 variant c.982C>T lies in exon 9, is present at a frequency of 0.00001961 with no homozygotes in gnomAD version 2.1.1 (control subjects) (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans [published correction appears in Nature 2021; 590:E53] [published correction appears in Nature 2021; 597:E3–E4].
      ), and causes missense change p.(R328W) (Figure S2C), predicted by PolyPhen-2 to be possibly damaging and by SIFT as deleterious (
      • Adzhubei I.
      • Jordan D.M.
      • Sunyaev S.R.
      Predicting functional effect of human missense mutations using PolyPhen-2.
      ,
      • Ng P.C.
      • Henikoff S.
      SIFT: Predicting amino acid changes that affect protein function.
      ) (Table S1). In a structural model of ANKRD2, the p.(R328W) variant appears to change the general conformation of the protein (Figure S2E). Both variants were absent from 50 ethnically matched Omani control DNAs.
      Table 2Mutations Identified in PDZD8
      FamilyEthnicityGenotypePDZD8 ModificationNucleotide ChangeFrequency in gnomADCADD Score
      AOmaniHomozygousp.(S733∗)c.2197_2200del035
      BEmiratiHomozygousp.(Y298∗)c.894C>G037
      Nucleotide and residue numbering are based on NM_173791.5.
      CADD, Combined Annotation Dependent Depletion.
      Because very little ANKRD2 (UniProtKB: Q9GZV1) is detected in the human brain (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ) and primary immunodeficiency is caused by missense changes in the gene (
      • Smedley D.
      • Smith K.R.
      • Martin A.
      • Thomas E.A.
      • McDonagh E.M.
      • et al.
      100,000 Genomes Project Pilot Investigators
      100,000 Genomes pilot on rare-disease diagnosis in health care—Preliminary report.
      ), the p.(R328W) variant appears unlikely to be responsible for ID in family A. However, because ANKRD2 is upregulated in congenital myopathies (
      • Nakada C.
      • Tsukamoto Y.
      • Oka A.
      • Nonaka I.
      • Sato K.
      • Mori S.
      • et al.
      Altered expression of ARPP protein in skeletal muscles of patients with muscular dystrophy, congenital myopathy and spinal muscular atrophy.
      ), p.(R328W) homozygosity may contribute to myopathy in family A.
      In family B, WES of the affected sibling (B.IV.2), with filtering for predicted pathogenic variants and segregation analysis, revealed a homozygous nonsense variant in PDZD8 exon 2 [NM_173791: c.894C>G; p.(Y298∗)] (Figure 1D and Table 2) as the most likely cause of his condition. The p.(Y298∗) variant is absent from gnomAD, and no other variants that potentially explain the phenotype were identified in his exome. gnomAD control datasets list five other predicted loss-of-function variants in PDZD8, none homozygous, and constraint metrics indicate that PDZD8 is extremely intolerant to loss-of-function variation (
      • Karczewski K.J.
      • Francioli L.C.
      • Tiao G.
      • Cummings B.B.
      • Alföldi J.
      • Wang Q.
      • et al.
      The mutational constraint spectrum quantified from variation in 141,456 humans [published correction appears in Nature 2021; 590:E53] [published correction appears in Nature 2021; 597:E3–E4].
      ).
      PDZD8 encodes an integral ER protein (UniProtKB: Q8NEN9) anchored to the membrane by an N-terminal TM helical domain (2–24 aa), which is followed by a synaptotagmin-like mitochondrial lipid-binding domain (91–294 aa), a PDZ (PSD-95/DlgA/ZO-1-like) domain (366–449 aa), a proline-rich region (551–626 aa), a C1 (phorbol-ester/diacylglycerol binding) domain (840–891 aa), and a coiled-coil domain (1028–1063 aa) (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ,
      UniProt Consortium
      UniProt: The universal protein Knowledgebase in 2021.
      ). If the p.(Y298∗) and p.(S733∗) variants evade nonsense-mediated messenger RNA (mRNA) decay (NMD) (
      • Supek F.
      • Lehner B.
      • Lindeboom R.G.H.
      To NMD or not to NMD: Nonsense-mediated mRNA decay in cancer and other genetic diseases.
      ), truncated PDZD8 proteins lacking 857 (p.Y298∗) or 422 (p.S733∗) C-terminal amino acids would be produced (Figure 1E and Figure S3). PDZD8 is highly expressed throughout the human brain (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ), including all subclasses of GABAergic (gamma-aminobutyric acidergic) and glutamatergic neurons in the adult primary motor cortex (
      • Bakken T.E.
      • Jorstad N.L.
      • Hu Q.
      • Lake B.B.
      • Tian W.
      • Kalmbach B.E.
      • et al.
      Comparative cellular analysis of motor cortex in human, marmoset and mouse.
      ) (Figure S4). Our analysis of bulk RNA sequencing data from the BrainSpan project (
      • Miller J.A.
      • Ding S.L.
      • Sunkin S.M.
      • Smith K.A.
      • Ng L.
      • Szafer A.
      • et al.
      Transcriptional landscape of the prenatal human brain.
      ) revealed that PDZD8 expression is relatively stable from 8 weeks after conception to early adulthood (23 years) across regions of the developing human brain (Figure S5), suggesting a role in neurodevelopment, making PDZD8 a strong candidate for involvement in ID.

      Long-term Memory Deficit in Drosophila Knockdown Model

      To assess PDZD8 function in cognition, we targeted the orthologous gene in D. melanogaster. The PDZD8 ortholog, CG10362 (FlyBase ID: FBgn0030358), encodes a 1037 aa protein (LD34222p; NP_572771.1; UniProtKB: Q9VYR9) that has a similar domain structure but relatively low amino acid conservation compared with human PDZD8 (24% identity). Drosophila PDZD8 has an N-terminal TM domain followed by a synaptotagmin-like mitochondrial lipid-binding domain, a PDZ domain, and a C1 domain but, unlike mammalian PDZD8, lacks a C-terminal coiled-coil domain (Figure 1E) (
      • Lee I.
      • Hong W.
      Diverse membrane-associated proteins contain a novel SMP domain.
      ). In adult flies, CG10362 expression is enriched in head, eye, brain, and thoracico-abdominal ganglion (noncephalic central nervous system) tissue (
      • Leader D.P.
      • Krause S.A.
      • Pandit A.
      • Davies S.A.
      • Dow J.A.T.
      FlyAtlas 2: A new version of the Drosophila melanogaster expression atlas with RNA-Seq, miRNA-Seq and sex-specific data.
      ). RNA interference–mediated knockdown (KD) of CG10362 reduced the level of the target CG10362 transcript by 56.32 ± 9.20% in adult Act5C-Gal4 x UAS-RNAi F1 (CG10362-KD) males upon comparison with UAS-RNAi and Act5C-Gal4 control animals (Figure 2A).
      Figure thumbnail gr2
      Figure 2Associative learning and memory in CG10362 KD flies. (A) Expression of CG10362 in four pools of 8 to 10 whole adult CG10362-KD (KD: 0.4417 ± 0.09643), UAS-RNAi (UAS: 1.006 ± 0.04253), and Act5C-Gal4 (Gal4: 1.001 ± 0.04712) male flies (one-way analysis of variance: F2,9 = 23.68, p < .0001; post hoc Bonferroni’s correction, Gal4 vs. UAS: p = 1.0, KD vs. UAS: p = .001, KD vs. Gal4: p = .001). (B) Aversive olfactory conditioning assay memory indices 30 seconds (learning) after training of KD (n = 8; 0.2393 ± 0.0442), UAS (n = 8; 0.35 ± 0.0634), and Gal4 (n = 7; 0.2024 ± 0.043) flies (one-way analysis of variance: F2,20 = 2.189, p = .1382) and 30 minutes (short-term memory) after training of KD (n = 7; 0.2405 ± 0.0956), UAS (n = 7; 0.2969 ± 0.0394), and Gal4 (n = 7; 0.2161 ± 0.0512) flies (Kruskal-Wallis: χ22 = 1.2764, p = .5282). (C) Courtship conditioning assay memory indices immediately (0 hours) after training of KD (n = 17; 0.4327 ± 0.1782), UAS (n = 20; 0.3493 ± 0.0994), and Gal4 (n = 18; 0.3169 ± 0.0772) flies (Kruskal-Wallis: χ22 = 0.8324, p = .6595); 30 minutes after training of KD (n = 22; 0.4868 ± 0.1085), UAS (n = 24; 0.5326 ± 0.1542), and Gal4 (n = 19; 0.5144 ± 0.1067) flies (Kruskal-Wallis: χ22 = 0.8672, p = .6482); and 48 hours after training of KD (n = 33; 1.2102 ± 0.0902), UAS (n = 27; 0.7301 ± 0.0786), and Gal4 (n = 25; 0.8123 ± 0.0669) flies (one-way analysis of variance: F2,82 = 10.52, p < .0001; post hoc Bonferroni’s correction, Gal4 vs. UAS: p = 1.0, KD vs. UAS: p = < .001, KD vs. Gal4: p = .003). Above dotted line (1.0) indicates no memory. Data are plotted as mean ± SEM. ∗∗p < .01 vs. controls; ##p < .01 vs. Gal4; ∗∗∗p < .001 vs. UAS. KD, knockdown; UAS, upstream activation sequence.
      In the aversive olfactory conditioning assay, the ability of CG10362-KD flies to memorize a novel association between an odor and mechanical shock for 30 seconds or 30 minutes was unaltered (Figure 2B). In the courtship conditioning assay, CG10362-KD males demonstrated reduced suppression of courtship behavior 48 hours, but not immediately or 30 minutes, after exposure to a premated female (Figure 2C). Unconditioned CG10362-KD males displayed robust courting behavior toward virgin females (data not shown). These results suggest that KD of CG10362 impairs long-term courtship-based memory but does not affect learning, short-term memory, or basal sexual behavior.

      Pdzd8tm1b Mice Exhibit Restricted Growth and Brain Structural Alterations

      To gain further insight to the cognitive effects of PDZD8 disruption, we studied the pre-existing Pdzd8tm1b mouse line, generated in the EUCOMM (European Conditional Mouse Mutagenesis) program (
      • Friedel R.H.
      • Seisenberger C.
      • Kaloff C.
      • Wurst W.
      EUCOMM—The European conditional mouse mutagenesis program.
      ), which harbors the mutation F333Nfs1∗ that closely models the PTCs identified in families A and B (Figure 1E). The mouse ortholog, Pdzd8, encodes a 1147 aa protein (UniProtKB: B9EJ80) that has 87% aa conservation with, and a similar domain structure to, human PDZD8. Real-time quantitative reverse transcription polymerase chain reaction confirmed the absence of Pdzd8 mRNA including exon 3 in Pdzd8tm1b mouse brain (Figure S6). Western blotting using an antibody to PDZD8, with an epitope between the C-terminal C1 and coiled-coil domains (Figure S3), detected a ∼140-kDa protein in WT mouse brain, which was 63.24 ± 3.84% less abundant in heterozygous samples and absent from Pdzd8tm1b samples (Figure S7), confirming the loss of full-length PDZD8 in Pdzd8tm1b mice.
      When intercrossing heterozygotes, Pdzd8tm1b pups were weaned at rates 36% and 48% lower than the expected Mendelian genotypic ratio in two separate colonies. Surviving Pdzd8tm1b mice appeared healthy but had a lower body weight and growth rate between 4 and 16 weeks of age (Figure S8A–D). Soft tissue mass (Figure S8E, F) and body length (Figure S8G, H) were reduced in 14-week-old Pdzd8tm1b mice. Structural magnetic resonance imaging to identify changes in brain morphology revealed a 7.07 ± 0.74% decrease in total brain volume in 16-week-old Pdzd8tm1b mice compared with WT littermates (Figure 3A). To assess differences in specific brain regions, the volume of each region was normalized to absolute brain volume. Pdzd8tm1b mice showed an increased relative volume of the cerebellum, olfactory bulb, hippocampus, and retrosplenial cortex (Figure 3B–E) but a decreased relative volume of the thalamus, pallidum, superior colliculus, and corpus callosum (Figure 3F–I), indicative of brain structural alterations. Representative structural images for Pdzd8tm1b and WT mice of each sex are shown in Figure S9.
      Figure thumbnail gr3
      Figure 3Voxelwise volumetric differences in whole brain and specific brain regions in Pdzd8tm1b mice determined by high-resolution structural magnetic resonance imaging. Significant differences in volume between Pdzd8tm1b (n = 32 [10 males, 22 females]) and WT (n = 17 [7 males, 10 females]) are indicated by red (increased volume) and blue (reduced volume) contour shading on two-dimensional coronal slice images of the brain. (A) Absolute brain volume (mm3). (B) Cerebellum: relative volume (% total brain volume). (C) Olfactory bulb: relative volume (% total brain volume). (D) Hippocampus: relative volume (% total brain volume). (E) Retrosplenial cortex: relative volume (% total brain volume). (F) Thalamus: relative volume (% total brain volume). (G) Pallidum: relative volume (% total brain volume). (H) Superior colliculus: relative volume (% total brain volume). (I) Corpus callosum: relative volume (% total brain volume). ∗∗∗∗p < .0001 vs. WT. WT, wild-type.

      Pdzd8tm1b Mice Exhibit Spontaneous Stereotypies and Decreased Anxiety-like Behavior

      In the home cage, adult Pdzd8tm1b mice frequently showed spontaneous repetitive hindlimb jumping behavior (Figure S10A), typically preceded by rearing against the wall, when housed in a mixed-genotype group (Video S1) or alone (Video S2). This stereotyped motor behavior was not observed in WT littermates.
      To assess Pdzd8tm1b mouse behavior in more detail, we observed mice in the open field test. In the novel arena, the total distances traveled were comparable between Pdzd8tm1b and WT littermates (Figure S10B), but the ambulatory activity of Pdzd8tm1b mice decreased more slowly over the course of the 1-hour test (Figure 4A, B), indicative of reduced habituation (
      • Bolivar V.J.
      • Caldarone B.J.
      • Reilly A.A.
      • Flaherty L.
      Habituation of activity in an open field: A survey of inbred strains and F1 hybrids.
      ,
      • Gallitano-Mendel A.
      • Izumi Y.
      • Tokuda K.
      • Zorumski C.F.
      • Howell M.P.
      • Muglia L.J.
      • et al.
      The immediate early gene early growth response gene 3 mediates adaptation to stress and novelty.
      ). Pdzd8tm1b mice had more entries to (Figure 4C), and spent more time (Figure 4D) and traveled further in (Figure S10C), the center of the arena (Figure S10D), suggestive of less anxiety-like behavior. Pdzd8tm1b mice also exhibited repetitive jumping behavior in the open field (Figure 4E–G).
      Figure thumbnail gr4
      Figure 4Behavioral differences of Pdzd8tm1b mice in OF and EPM. (A) Distance traveled (m) by Pdzd8tm1b (n = 12) and WT (n = 14) mice in 10-minute intervals in OF, with lines of best fit shown (two-way repeated-measures analysis of variance, genotype: F1,24 = 3.037, p = .094; time: F2.44,58.65 = 23.17, p < .0001; interaction: F2.44,58.65 = 3.795, p = .021). (B) Slope of habituation curve of Pdzd8tm1b (−0.1625 ± 0.07680) and WT (−0.3727 ± 0.04413) mice (independent t test: t24 = 2.458, p = .028). (C) Number of entries by Pdzd8tm1b (92.67 ± 11.28) and WT (58.36 ± 8.195) mice to OF inner zone (independent t test: t24 = −2.508, p = .019). (D) Time (s) spent by Pdzd8tm1b (104.00 ± 21.44) and WT (51.69 ± 8.965) mice in OF inner zone (independent t test: t24 = −2.710, p = .012). (E) Representative image of hindlimb jumping by Pdzd8tm1b mouse in OF. (F) Number of jumps by Pdzd8tm1b (n = 11; 165.9 ± 92.78) and WT (n = 12; 12.58 ± 6.289) mice in OF (Mann-Whitney U test: U = 97.00, p = .051). (G) Percentage of Pdzd8tm1b and WT mice making more than 10 jumps per 10-minute interval in OF (Fisher’s exact test, 10–20 min: p = .037; 20–30 min: p = .037; 50–60 min: p = .037). (H) Number of entries to closed arms by Pdzd8tm1b (17.0 ± 1.317) and WT (18.06 ± 1.184) mice in EPM (independent t test: t32 = 0.597, p = .5541). (I) Number of entries to open arms by Pdzd8tm1b (7.5 ± 1.258) and WT (4.056 ± 0.5686) mice in EPM (Welch’s t test: t32 = −2.494, p = .021). (J) Number of head dips by Pdzd8tm1b (21.81 ± 2.91) and WT (12.33 ± 1.06) mice in EPM (Welch’s t test: t32 = −3.061, p = .006). (K) Total distance traveled (m) by Pdzd8tm1b (n = 16; 12.69 ± 1.27) and WT (n = 18; 11.60 ± 0.79) mice in EPM (Mann-Whitney U test: U = 133, p = .720). Data are plotted as mean ± SEM. ∗p < .05; ∗∗p < .01 vs. WT. EPM, elevated plus maze; OF, open field; WT, wild-type.
      In the elevated plus maze, Pdzd8tm1b mice exhibited a greater frequency of open arm entries (Figure 4I) and head dips (protruding the head over the edge of an open arm and down toward the floor) (Figure 4J) than WT littermates, suggesting decreased anxiety and increased exploration (
      • Rodgers R.J.
      • Johnson N.J.
      Factor analysis of spatiotemporal and ethological measures in the murine elevated plus-maze test of anxiety.
      ,
      • Griebel G.
      • Rodgers R.J.
      • Perrault G.
      • Sanger D.J.
      Risk assessment behaviour: Evaluation of utility in the study of 5-HT-related drugs in the rat elevated plus-maze test.
      ,
      • Weiss S.M.
      • Wadsworth G.
      • Fletcher A.
      • Dourish C.T.
      Utility of ethological analysis to overcome locomotor confounds in elevated maze models of anxiety.
      ).

      Long-term Spatial Memory and TBS-Induced LTP Are Impaired in Pdzd8tm1b Mice

      In the Y maze, Pdzd8tm1b and WT mice showed roughly equivalent levels of spontaneous alternation, a measure of spatial working memory (Figure S11D). In the Barnes maze, the performance levels of Pdzd8tm1b and WT mice were similar over the 4 days of training (Figure 5A–C and Figure S11E, F), demonstrating intact spatial learning. During the probe trial 24 hours after the last training trial, Pdzd8tm1b mice spent less time in the target quadrant, target sector, and target hole annulus and had a lower probability of entering the target hole than WT mice (Figure 5D–G), indicative of a hippocampal-dependent spatial memory impairment in Pdzd8tm1b mice.
      Figure thumbnail gr5
      Figure 5Performance of Pdzd8tm1b (n = 15) and WT (n = 10) mice in Barnes maze. (A) Latency (s) to first head entry to escape hole (Friedman’s analysis of variance, Pdzd8tm1b: χ23 = 19.88, p < .0001; WT: χ23 = 21.30, p < .0001. Mann-Whitney U test, day 1: U = 79.50, p = .80; day 2: U = 85.50, p = .56; day 3: U = 78.00, p = .868; day 4: U = 98.00, p = .216). (B) Primary path length (m) (Friedman’s analysis of variance, Pdzd8tm1b: χ23 = 11.32, p = .01; WT: χ23 = 23.88, p < .0001. Mann-Whitney U test, day 1: U = 60.00, p = .42; day 2: U = 76.00, p = 1.00; day 3: U = 83.00, p = .68; day 4: U = 92.00, p = .367). (C) Number of errors. (Friedman’s analysis of variance, Pdzd8tm1b: χ23 = 10.50, p = .015; WT: χ23 = 9.39, p = .024. Mann-Whitney U test, day 1: U = 48.50, p = .144; day 2: U = 72.00, p = .892; day 3: U = 67.50, p = .683; day 4: U = 94.00, p = .311). (D) Time (s) spent by Pdzd8tm1b (39.76 ± 4.983) and WT (53.45 ± 3.273) mice in target quadrant (Welch’s t test: t22.24 = 2.296, p = .031. One-sample t test, Pdzd8tm1b vs. chance [20]: t14 = 3.965, p = .0014; WT vs. chance [20]: t9 = 10.22, p < .0001). (E) Time (s) spent by Pdzd8tm1b (11.39 ± 2.088) and WT (21.96 ± 2.579) mice in target sector (independent t test: t23 = 3.223, p = .004. One-sample t test, Pdzd8tm1b vs. chance [4]: t14 = 3.540, p = .0033; WT vs. chance [4]: t9 = 6.965, p < .0001). (F) Time (s) spent by Pdzd8tm1b (6.587 ± 1.643) and WT (12.35 ± 2.3) mice within target hole annulus (Mann-Whitney U test: U = 36.00, p = .031). (G) Left, entry probability (%) of Pdzd8tm1b (13.64 ± 2.359) and WT (27.8 ± 3.086) mice into the target hole annulus (independent t test: t23 = 3.69, p = .001). Right, heat maps of mean entry probability (%) of Pdzd8tm1b (right) and WT (left) mice. Data are plotted as mean ± SEM. ∗p < .05; ∗∗p < .01 vs. WT. T, target sector; WT, wild-type.
      We next used electrophysiology to examine synaptic plasticity in acute hippocampal slices from Pdzd8tm1b mice. Experimentally induced LTP of synaptic transmission is a widely accepted model of synaptic plasticity that involves molecular and cellular processes engaged during the biological consolidation of memories (
      • Bliss T.V.
      • Collingridge G.L.
      A synaptic model of memory: Long-term potentiation in the hippocampus.
      ). Three different stimulation protocols to induce LTP at Schaffer collateral–CA1 stratum radiatum synapses were used: 3× TBS (a maximal stimulation that induces saturated LTP), 1× TBS, and 1× HFS (submaximal). The magnitudes of LTP evoked by 1× TBS and 1× HFS at 30 minutes after stimulation were comparable between Pdzd8tm1b and WT slices (Figure 6A, C). However, the LTP evoked by 3× TBS was diminished in Pdzd8tm1b compared with WT slices (Figure 6B), indicating that Pdzd8tm1b mice have a specific deficit in 3× TBS-evoked LTP and are not capable of generating as much synaptic potentiation as WT mice.
      Figure thumbnail gr6
      Figure 6Analysis of hippocampal (long-term potentiation) in Pdzd8tm1b mice. (A) Normalized change in fEPSP (% baseline) induced by 1× TBS in Pdzd8tm1b (131 ± 4%; n = 11) and WT (136 ± 6%; n = 8) mice. (B) Normalized change in fEPSP (% baseline) induced by 3× TBS in Pdzd8tm1b (131 ± 4%; n = 15) and WT (156 ± 7%; n = 16) mice. Scale bars = 0.3 mV and 10 ms in (A) and (B). (C) Normalized change in fEPSP (% baseline) induced by 1× HFS in Pdzd8tm1b (145 ± 7%; n = 10) and WT (135 ± 6%; n = 12) mice. Insets: representative traces before (WT, light blue; Pdzd8tm1b, pink) and after (WT, blue; Pdzd8tm1b, red) the induction protocol. Scale bars = 0.2 mV and 10 ms. (D) Facilitation of fEPSP (% baseline) at 30 minutes after 1× TBS, 3× TBS, and 1× HFS. (E) Paired-pulse ratio of Pdzd8tm1b (1.48 ± 0.03; n = 43) and WT (1.63 ± 0.04; n = 39) mice with 50-ms stimulus interval. Representative traces of WT (blue) and Pdzd8tm1b (red) slices. Scale bars = 0.2 mV and 100 ms. Data are plotted as mean ± SEM. ∗p < .05 vs. WT. fEPSP, field excitatory postsynaptic potential; HFS, high-frequency stimulation; TBS, theta burst stimulation; WT, wild-type.
      To evaluate presynaptic short-term plasticity at Schaffer collateral–CA1 synapses in Pdzd8tm1b and WT slices, we used paired-pulse stimulation with a 50-ms interval between the first and second pulses. Both genotypes exhibited paired-pulse facilitation of excitatory synaptic transmission (Figure 6E), postulated to result from a transient increase in Ca2+ levels in the presynaptic terminal. However, the lower paired-pulse facilitation of Pdzd8tm1b slices suggests a higher initial probability of neurotransmitter release associated with the first pulse or reduced residual Ca2+ resulting from altered Ca2+ uptake (
      • Zucker R.S.
      • Regehr W.G.
      Short-term synaptic plasticity.
      ).

      Discussion

      We have identified homozygous PTC variants p.(Y298∗) and p.(S733∗) in PDZD8 cosegregating with syndromic ID in two independent consanguineous families from the Arabian Peninsula. Such mutations are by default often considered loss-of-function events for the protein-coding genes that harbor them, in part because of the assumption that the PTC-containing mRNA is degraded by NMD (
      • Supek F.
      • Lehner B.
      • Lindeboom R.G.H.
      To NMD or not to NMD: Nonsense-mediated mRNA decay in cancer and other genetic diseases.
      ). Because NMD is less efficient for PTCs in the last exon (
      • Cirulli E.T.
      • Heinzen E.L.
      • Dietrich F.S.
      • Shianna K.V.
      • Singh A.
      • Maia J.M.
      • et al.
      A whole-genome analysis of premature termination codons.
      ), it is possible that p.(S733∗) located in the last exon of PDZD8 may evade PTC detection and mRNA degradation. If a PTC-bearing allele does escape detection by NMD, protein translation would stop prematurely, and thus no functional full-length PDZD8 protein would be produced.
      The lack of PDZD8 protein in human blood limits our ability to detect truncated PDZD8 proteins (
      • Uhlén M.
      • Fagerberg L.
      • Hallström B.M.
      • Lindskog C.
      • Oksvold P.
      • Mardinoglu A.
      • et al.
      Proteomics. Tissue-based map of the human proteome.
      ). If produced, truncated PDZD8 would retain the N-terminal TM domain, anchoring the protein to the ER, and the synaptotagmin-like mitochondrial lipid-binding domain involved in dimerization (
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ). However, it would lack C-terminal regions involved in interaction with another ER TM protein, protrudin (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ); the phospholipids phosphatidylserine and phosphatidylinositol 4-phosphate, associated with the late endosome/lysosome membrane (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Hammond G.R.V.
      • Machner M.P.
      • Balla T.
      A novel probe for phosphatidylinositol 4-phosphate reveals multiple pools beyond the Golgi.
      ); and the late endosomal Rab GTPase, Rab-7a (
      • Elbaz-Alon Y.
      • Guo Y.
      • Segev N.
      • Harel M.
      • Quinnell D.E.
      • Geiger T.
      • et al.
      PDZD8 interacts with Protrudin and Rab7 at ER-late endosome membrane contact sites associated with mitochondria.
      ,
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ,
      • Guillén-Samander A.
      • Bian X.
      • De Camilli P.
      PDZD8 mediates a Rab7-dependent interaction of the ER with late endosomes and lysosomes.
      ). As an in-frame PTC, p.(Y298∗) is potentially amenable to novel nonsense suppression therapies aimed at suppressing PTCs to restore deficient protein function (
      • Martins-Dias P.
      • Romão L.
      Nonsense suppression therapies in human genetic diseases.
      ).
      To our knowledge, there are no other reports of disease-causing mutations in PDZD8. Although PDZD8 is one of multiple genes occasionally hemizygously lost in distal 10q deletion syndrome, in which ID and dysmorphic features are common (
      • Yatsenko S.A.
      • Kruer M.C.
      • Bader P.I.
      • Corzo D.
      • Schuette J.
      • Keegan C.E.
      • et al.
      Identification of critical regions for clinical features of distal 10q deletion syndrome.
      ,
      • Nandyal R.
      • Hagan S.
      • Sandhu T.
      Neonate with 10q interstitial deletion within the long arm of chromosome 10–A case report and literature review.
      ), our finding that syndromic ID is absent from heterozygotes in families A and B suggests that PDZD8 haploinsufficiency is unlikely to have a major impact. A mutation in protrudin (ZFYVE27) has previously been identified in spastic paraplegia (
      • Mannan A.U.
      • Krawen P.
      • Sauter S.M.
      • Boehm J.
      • Chronowska A.
      • Paulus W.
      • et al.
      ZFYVE27 (SPG33), a novel spastin-binding protein, is mutated in hereditary spastic paraplegia.
      ), while mutations in Rab-7a (RAB7A) have been identified in Charcot-Marie-Tooth type 2B neuropathy (
      • Verhoeven K.
      • De Jonghe P.
      • Coen K.
      • Verpoorten N.
      • Auer-Grumbach M.
      • Kwon J.M.
      • et al.
      Mutations in the small GTP-ase late endosomal protein RAB7 cause Charcot-Marie-Tooth type 2B neuropathy.
      ,
      • Houlden H.
      • King R.H.M.
      • Muddle J.R.
      • Warner T.T.
      • Reilly M.M.
      • Orrell R.W.
      • Ginsberg L.
      A novel RAB7 mutation associated with ulcero-mutilating neuropathy.
      ,
      • Meggouh F.
      • Bienfait H.M.E.
      • Weterman M.A.J.
      • de Visser M.
      • Baas F.
      Charcot-Marie-Tooth disease due to a de novo mutation of the RAB7 gene.
      ,
      • Rotthier A.
      • Baets J.
      • De Vriendt E.
      • Jacobs A.
      • Auer-Grumbach M.
      • Lévy N.
      • et al.
      Genes for hereditary sensory and autonomic neuropathies: A genotype-phenotype correlation.
      ).
      To assess PDZD8 function in cognition, we undertook a comparative study using targeted interference of PDZD8 orthologs in two model organisms. CG10362-KD fruit flies with KD of the PDZD8 ortholog showed intact learning and short-term memory in the aversive olfactory conditioning and courtship conditioning assays. However, when tested 48 hours after training in the courtship conditioning assay, CG10362-KD males showed deficient long-term courtship-based memory, consistent with interference of PDZD8 impairing long-term memory formation or recall.
      Pdzd8tm1b mice with a PTC (p.F333Nfs1∗) exhibited decreased preweaning viability. Surviving Pdzd8tm1b mice showed spontaneous repetitive hindlimb jumping, a stereotyped motor behavior with potential relevance to lower-order human motor stereotypies, such as hand flapping, common in ASD (
      American Psychiatric Association
      Diagnostic and Statistical Manual of Mental Disorders.
      ). High levels of jumping behavior have been reported in Shank2 null (
      • Won H.
      • Lee H.R.
      • Gee H.Y.
      • Mah W.
      • Kim J.I.
      • Lee J.
      • et al.
      Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.
      ), Sh3rf2 haploinsufficient (
      • Wang S.
      • Tan N.
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      Sh3rf2 haploinsufficiency leads to unilateral neuronal development deficits and autistic-like behaviors in mice.
      ), Camk2a-E183V (
      • Stephenson J.R.
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      • Shonesy B.C.
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      A novel human CAMK2A mutation disrupts dendritic morphology and synaptic transmission, and causes ASD-related behaviors.
      ), and Nlgn2 overexpression (
      • Hines R.M.
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      • Steenland H.
      • Mansour S.
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      • et al.
      Synaptic imbalance, stereotypies, and impaired social interactions in mice with altered neuroligin 2 expression.
      ) mice that model genetic risk factors for ASD and in the C58 inbred strain described as a mouse model of autism (
      • Silverman J.L.
      • Smith D.G.
      • Sukoff Rizzo S.J.
      • Karras M.N.
      • Turner S.M.
      • Tolu S.S.
      • et al.
      Negative allosteric modulation of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse models of autism.
      ). Approximately 10% of children with ID show or develop autistic symptoms (
      • Oeseburg B.
      • Dijkstra G.J.
      • Groothoff J.W.
      • Reijneveld S.A.
      • Jansen D.E.M.C.
      Prevalence of chronic health conditions in children with intellectual disability: A systematic literature review.
      ), including all affected individuals in families A and B.
      Across the 1-hour open field test, Pdzd8tm1b mice demonstrated reduced habituation—a decrease in response to a stimulus as it becomes familiar—postulated to reflect a deficit in information acquisition (
      • Platel A.
      • Porsolt R.D.
      Habituation of exploratory activity in mice: A screening test for memory enhancing drugs.
      ). In the Barnes maze, Pdzd8tm1b mice demonstrated reduced memory of the escape hole location in the probe trial, indicative of long-term spatial memory impairment. Our finding that interference of PDZD8 orthologs resulted in long-term memory deficits in mice and fruit flies provides cross-species substantive evidence linking PDZD8 disruption and cognitive impairment.
      In parallel with their hippocampal-dependent spatial memory impairment, Pdzd8tm1b mice showed diminished LTP evoked by 3× TBS, a maximal induction protocol, in acute hippocampal slices, suggesting that LTP may saturate at lower levels in Pdzd8tm1b mice. The magnitudes of LTP evoked by the 1× TBS and 1× HFS protocols were, however, not different between genotypes. This suggests that Pdzd8tm1b mice do not have a global impairment in synaptic function but a subtle hippocampal disruption revealed only when the number of TBS trains is increased or performance on a cognitively demanding task, such as the Barnes maze, is assessed. The reduction in synaptic plasticity at 4 to 6 weeks suggests that plasticity deficits may occur throughout development, and thus learning in the adult may be affected via cumulative synaptic deficits as well as directly.
      The ER constitutes a large and important source of Ca2+ for various neuronal signaling processes. Ca2+ is mobilized from intracellular ER stores upon activation of ryanodine receptors enriched in the dentate gyrus and CA3/4 fields of the hippocampus and/or inositol trisphosphate (IP3) receptors enriched in hippocampal CA1 pyramidal cells (
      • Sharp A.H.
      • McPherson P.S.
      • Dawson T.M.
      • Aoki C.
      • Campbell K.P.
      • Snyder S.H.
      Differential immunohistochemical localization of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channels in rat brain.
      ,
      • Maggio N.
      • Vlachos A.
      Synaptic plasticity at the interface of health and disease: New insights on the role of endoplasmic reticulum intracellular calcium stores.
      ). Synaptic plasticity in LTP induction paradigms comparable to our 3× TBS protocol is dependent on the activation of group I metabotropic glutamate receptors (mGluR1 and mGluR5), which results in the stimulation of phospholipase C, leading to IP3-mediated Ca2+ mobilization from the ER (
      • Raymond C.R.
      • Thompson V.L.
      • Tate W.P.
      • Abraham W.C.
      Metabotropic glutamate receptors trigger homosynaptic protein synthesis to prolong long-term potentiation.
      ,
      • Raymond C.R.
      • Redman S.J.
      Different calcium sources are narrowly tuned to the induction of different forms of LTP.
      ,
      • Raymond C.R.
      • Redman S.J.
      Spatial segregation of neuronal calcium signals encodes different forms of LTP in rat hippocampus.
      ,
      • Shalin S.C.
      • Hernandez C.M.
      • Dougherty M.K.
      • Morrison D.K.
      • Sweatt J.D.
      Kinase suppressor of Ras1 compartmentalizes hippocampal signal transduction and subserves synaptic plasticity and memory formation.
      ). Ca2+ mobilized from ER stores transiently reaches concentrations high enough to open the mitochondrial Ca2+ uniporter at ER-mitochondria contacts, promoting rapid mitochondrial Ca2+ import (
      • Patron M.
      • Checchetto V.
      • Raffaello A.
      • Teardo E.
      • Vecellio Reane D.
      • Mantoan M.
      • et al.
      MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity.
      ).
      In mouse cortical layer II/III pyramidal neurons, synaptic stimulation was shown to induce robust ER Ca2+ release and mitochondrial Ca2+ uptake in proximal dendrites (
      • Hirabayashi Y.
      • Kwon S.K.
      • Paek H.
      • Pernice W.M.
      • Paul M.A.
      • Lee J.
      • et al.
      ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.
      ), in which IP3 receptors are found (
      • Sharp A.H.
      • McPherson P.S.
      • Dawson T.M.
      • Aoki C.
      • Campbell K.P.
      • Snyder S.H.
      Differential immunohistochemical localization of inositol 1,4,5-trisphosphate- and ryanodine-sensitive Ca2+ release channels in rat brain.
      ). This effect was abolished by antagonism of mGluR1, consistent with the known role of mGluR activation in triggering efficient Ca2+ release from ER stores (
      • Frenguelli B.G.
      • Potier B.
      • Slater N.T.
      • Alford S.
      • Collingridge G.L.
      Metabotropic glutamate receptors and calcium signalling in dendrites of hippocampal CA1 neurones.
      ). KD of Pdzd8 has been shown to decrease proximity of the ER and mitochondria (
      • Hertlein V.
      • Flores-Romero H.
      • Das K.K.
      • Fischer S.
      • Heunemann M.
      • Calleja-Felipe M.
      • et al.
      MERLIN: A novel BRET-based proximity biosensor for studying mitochondria-ER contact sites.
      ) and decrease mitochondrial Ca2+ import evoked by synaptic stimulation, leading to significantly elevated cytosolic Ca2+ levels despite unchanged ER Ca2+ release (
      • Hirabayashi Y.
      • Kwon S.K.
      • Paek H.
      • Pernice W.M.
      • Paul M.A.
      • Lee J.
      • et al.
      ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.
      ). The effect of this on Ca2+ buffering and adenosine triphosphate synthesis by mitochondria at the synapse is likely to compromise neuronal and synaptic functioning (
      • Datta S.
      • Jaiswal M.
      Mitochondrial calcium at the synapse.
      ). PDZD8 loss of function may have had similar effects following 3× TBS in Pdzd8tm1b hippocampal slices. Such an impairment of hippocampal neurophysiology that supports spatial memory in Pdzd8tm1b mice is consistent with the high expression of Pdzd8 in the mouse hippocampus (
      • Hirabayashi Y.
      • Kwon S.K.
      • Paek H.
      • Pernice W.M.
      • Paul M.A.
      • Lee J.
      • et al.
      ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.
      ,
      • Lein E.S.
      • Hawrylycz M.J.
      • Ao N.
      • Ayres M.
      • Bensinger A.
      • Bernard A.
      • et al.
      Genome-wide atlas of gene expression in the adult mouse brain.
      ). In common with other neurodevelopmental disorders, the cognitive impairment associated with PDZD8 disruption is therefore likely to represent a synaptopathy resulting from synaptic dysfunction (
      • Volk L.
      • Chiu S.L.
      • Sharma K.
      • Huganir R.L.
      Glutamate synapses in human cognitive disorders.
      ).
      Adult Pdzd8tm1b mice exhibited decreased absolute brain volume, likely related to their decreased body size, which affected the cerebrum but not the cerebellum and olfactory bulb. Normalizing the volume of each region to absolute brain volume revealed multiple regions with altered relative volumes in Pdzd8tm1b mice, including the corpus callosum and hippocampus. The relative reduction of the corpus callosum replicates the corpus callosal hypoplasia of the affected female (A.IV.5) in family A. The relative expansion of the hippocampus is comparable to that observed in other mouse models of neurodevelopmental disorders exhibiting cognitive deficits. The heterozygous Arid1b knockout mouse model of Coffin-Siris syndrome, a syndromic ID, exhibits body weight and growth rate deficits, reduced total brain volume but hippocampal enlargement, and deficits in novel object recognition (
      • Ellegood J.
      • Petkova S.P.
      • Kinman A.
      • Qiu L.R.
      • Adhikari A.
      • Wade A.A.
      • et al.
      Neuroanatomy and behavior in mice with a haploinsufficiency of AT-rich interactive domain 1B (ARID1B) throughout development.
      ). The heterozygous Chd8 mutant mouse model of ASD exhibits an increase in hippocampal volume that is correlated with deficits in contextual fear conditioning (
      • Gompers A.L.
      • Su-Feher L.
      • Ellegood J.
      • Copping N.A.
      • Riyadh M.A.
      • Stradleigh T.W.
      • et al.
      Germline Chd8 haploinsufficiency alters brain development in mouse.
      ). Deciphering how PDZD8 disruption affects the tightly orchestrated and intricate processes that determine brain structure will require additional studies.
      To summarize, PDZD8 is an ER TM protein required for the extraction of lipids from the ER to late endosomes and lysosomes (
      • Shirane M.
      • Wada M.
      • Morita K.
      • Hayashi N.
      • Kunimatsu R.
      • Matsumoto Y.
      • et al.
      Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation.
      ,
      • Gao Y.
      • Xiong J.
      • Chu Q.Z.
      • Ji W.K.
      PDZD8-mediated lipid transfer at contacts between the ER and late endosomes/lysosomes is required for neurite outgrowth.
      ) and mitochondrial Ca2+ uptake following synaptic transmission–induced Ca2+ release from ER stores (
      • Hirabayashi Y.
      • Kwon S.K.
      • Paek H.
      • Pernice W.M.
      • Paul M.A.
      • Lee J.
      • et al.
      ER-mitochondria tethering by PDZD8 regulates Ca2+ dynamics in mammalian neurons.
      ). Our data demonstrate the involvement of homozygous loss-of-function mutations in PDZD8 in syndromic ID. This knowledge will benefit affected families through genetic counseling and carrier screening and facilitate the genetic diagnosis of other patients. Disrupting the orthologous gene resulted in long-term memory deficits in fruit flies and brain structural alterations, long-term memory deficits, and impaired hippocampal neurophysiology in mice, replicating aspects of the human phenotype and demonstrating a critical role for PDZD8 in brain development and synaptic plasticity. We can use these models to decipher precisely how PDZD8 disruption affects neurodevelopment and synapse function, thus providing insight to the pathophysiological mechanism and potential treatment of this lifelong disability.

      Acknowledgments and Disclosures

      This study was supported by the Medical Research Council (Grant No. MR/R014736/1 [to CFI and SJC]), the Biotechnology and Biological Sciences Research Council (Grant Nos. BB/R019401/1 [to SJC] and BB/R002177/1 [to JRM and MAm]), the Wellcome Trust (Grant No. 101029/Z/1/Z [to JRM and MAm]), the National Centre for the Replacement, Refinement and Reduction of Animals in Research (Grant No. NC/S001719/1 [to AB and SJC]), a Ph.D. scholarship from the Oman Ministry of Higher Education (to AHA-A), a Ph.D. scholarship from the Emma Reid and Leslie Reid Scholarships and Fellowships Fund (to AP), an M.Sc. studentship from the Ethel and Gwynne Morgan Trust (to HWYN), and a summer internship from the British Association for Psychopharmacology (to KG).
      We thank both families who participated in this study. We thank Tim Munsey, Ben Slater, Georgia Hale, Katharine Wiltshire, Alix Stonehouse, Frances Moore, Zoe Hewitson, Amy Aspinall, Stephen Pickett, Shivali Kohli, and Tom Wainwright for technical assistance.
      RA-A and CB are employees of Centogene GmbH. All other authors report no biomedical financial interests or potential conflicts of interest.

      Supplementary Material

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